土壤中烟草根黑腐病菌分子检测技术研究
首发时间:2009-04-03
摘要:烟草根黑腐病菌是一种引起烟草根腐烂的土传病菌。基于形态学或生理特征检测该病菌的方法费时、费力并且需要广泛的真菌知识。rDNA的内转录间隔区的分子检测是一种新颖有效的物种鉴定方法。一对特异引物对Tb1/Tb2设计在两个内转录间隔区域,扩增片段大小为338bp。引物对对其他供试的土传病菌,如:烟草疫霉病菌(Phytophthora nicotianae),恶疫霉(Phytophthora cactorum),烟草白星病菌(Cercospora nicotianae),棉花立枯病病菌(Fusaricum oxysporium),棉花黄萎病菌(Verticillium dahiliae),欧文式杆菌(Erwinia carotovora),枯草芽孢杆菌(Bacillus subtilis),烟草青枯病菌(Ralstonia solanacearum)和分离培养的土壤优势菌均不能扩增出任何条带。利用10倍稀释的DNA作阳性标准品,荧光定量PCR灵敏度检测下限为10 fg/μL,土壤中分生孢子检测下限为20个/克土壤。人工接种土壤分析表明病菌孢子浓度和实时PCR阈值之间的线性相关性为0.93。
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Studies on the molecular detection of the fungi caused tobacco black root rot in soil
Abstract:Thielaviopsis basicola is a kind of soil-borne plant pathogens which is known to cause root-rot diseases in tobacco plants. The identification of this pathogen based on morphological or physiologcial characters is time-consuming and labour-intensive and requires comprehensive knowledge of fungi. Molecular analysis of the internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. A specific primer pair Tb1/Tb2, which was developed from the ITS 1 and 2 regions of the ribosomal RNA genes, gave a 338 bp amplicon with Thielaviopsis basicola. The primer pairs revealed no undesirable cross-reaction with a diverse test collection of isolates including other soil-borne pathogens, Phytophthora nicotianae, Phytophthora cactorum, Cercospora nicotianae, Fusaricum oxysporium, Verticillium dahiliae,Erwinia carotovora,Bacillus subtilis,Ralstonia solanacearum and three soil predominant pathogens which were isolated and cultivated. In real-time PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg μL -1 for Thielaviopsis basicola and 20 zoospores g-1 soil for artifially infested soil. The analyses of artificially infested soil showed that the concentration of pathogen propagules and the real-time PCR cycle threshold(Ct) was 0.93.
Keywords: internal transcribe spacer(ITS) real-time PCR Thielaviopsis basicola conidium
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